1. Prior Art
Many diseases occurring in humans and animals can be detected by the presence of foreign substances, particularly in the blood, which are specifically associated with the disease or condition. Tests for antigens or other such substances produced as a result of such diseases show great promise as a diagnostic tool for the early detection and treatment of the particular disease that produced the antigen or other substance. A procedure for the detection of such substances must be reliable, reproducible, and sensitive in order to constitute a practical diagnostic procedure for health care providers. In addition, any such procedure should be able to be carried out by persons of ordinary skill and training in laboratory procedures, and should be relatively fast and inexpensive to carry out.
For example, in the treatment of the various malignancies that afflict humans and animals, referred to generally as cancer, it is recognized that early detection is a key to effective treatment, especially as many therapeutic procedures are effective only in relatively early stages of the disease. In fact, virtually all known cancer treatments are not only more effective, but safer, in early stages of cancer. Far too many cases of cancer are only discovered too late for effective treatment.
Accordingly, there is a great need for reliable tests which can diagnose cancer at early stages. In this connection new tests and procedures are being developed to effect early diagnosis of the cancer.
One extremely desirable aspect of such a test is its ability either to detect all types of cancer generally, or to detect specific types of cancer, depending on the materials used. The former application of such a test is very important in mass screening of large patient populations, as would be done in routine checkups. In such mass screenings a test dependent on a particular type of cancer would not be desirable, as there are literally hundreds of types of cancer and a test that could spot only one or a few types of the disease is far too likely to miss many cases of caner. In general, these patients would present either no symptoms or vague generalized symptoms that could not be readily linked to a particular type of cancer, so there would be no basis for suspecting a particular type and administering a test specific for that type.
In contrast, once the presence of malignancy is known or strongly suspected, it would be desirable to have a test that could pinpoint the particular type of malignancy present. Such a test could add greatly to the efficacy of treatment, because many of the most effective cancer therapies, such as chemotherapy, are only effective against one type of cancer or at best, a narrow range of types, and the wrong chemotherapy can do more harm than good.
We have developed and reported one such test for the early detection of cancer in L. Cercek, B. Cercek, and C. I. V. Franklin, "Biophysical Differentiation Between Lymphocytes from Health Donors, Patients with Malignant Diseases and Other Disorders," Brit. J. Cancer 29, 345-352 (1974) and L. Cercek and B. Cercek, "Application of the Phenomenon of Changes in the Structuredness of Cytoplasmic Matrix (SCM) in the Diagnosis of Malignant Disorders: a Review," Europ. J. Cancer 13, 903-915 (1977).
Our SCM (structuredness of cytoplasmic matrix) test takes advantage of the fact that a subpopulation of the lymphocytes of a normal individual alters their internal structure in response to challenge by a mitogen such as phytohaemagglutinin (PHA), and does not respond to challenge by certain challenging agents, including certain cancer-associated antigens, while the lymphocytes of an individual with cancer respond in just the reverse way. In other words the same subpopulation of lymphocytes from cancer patients does not respond in the SCM test to challenge by a mitogen, but does respond strongly to challenge by a number of cancer-associated antigens, as well as by the synthetic peptides which are the subject of the present invention.
Depending on the substance used to challenge the lymphocytes (the challenging agent), the SCM test can be made to detect either cancer generally or a specific type of cancer. As will be detailed, the SCM test shows considerable promise in detecting early malignancies, and therefore, has potential clinical usefulness.
The changes seen in the SCM test are believed to reflect changes in the internal structure of the lymphocyte as the lymphocyte is activated for synthesis. These changes are seen as a decrease in the fluorescence polarization of the cells when polarized light is used to excite an extrinsic fluor, fluorescein, generated intracellularly by the hydrolysis of a nonfluorescent compound, fluorescein diacetate, which has been absorbed by the lymphocytes. It is necessary to add an extrinsic fluor since the intrinsic fluorescence of cellular components is too small to give results in this test. Therefore, all references to fluorescence polarization values herein are references to fluorescence polarization values obtained with an extrinsic fluor, preferably one generated intracellularly by enzymatic hydrolysis from a nonfluorogenic compound added to and absorbed by the cells.
When measuring fluorescence polarization in the SCM test, the important value to calculate is the net value of fluorescence polarization, "P", after correction is made for (i) the intrinsic fluorescence of the medium, (ii) any extracellular fluorescein present, and (iii) the unequal transmission of the two components of polarized light in the fluorescence polarization measurement apparatus. All references hereinafter to measurements of the fluorescence polarization are to measurements of the net value of the fluorescence polarization unless otherwise specified. Fluorescence polarization is a measure of intracellular rigidity; the greater the fluidity of intracellular mobility, the less the measured fluorescence polarization. As seen in the SCM test, the observed decrease in fluorescence polarization, as measured by a decrease in the P value, is thought to result mainly from changes in the conformation of the mitochondria, the energy-producing organelles of the cell. The changes in the mitochondria are believed to result from the contractions of the cristae or inner folds of the mitochondrial membrane. The SCM reflects the forces of interaction between macromolecules and small molecules such as water molecules, ions, adenosine triphosphate, and cyclic adenosine phosphate. Perturbations of these interactions results in changes in the SCM.
Not all lymphocytes respond positively in the SCM test; only about 10-20% of lymphocytes actually respond. Immunologically the SCM-responding lymphocytes are T-cell mononuclear leukocytes. These lymphocytes which do respond form a subpopulation which can be isolated by methods such as density gradient centrifugation. This subpopulation is referred to hereinafter as "potentially SCM-responding lymphocytes."
The SCM test is capable of responding to a relatively small quantity of malignant cells. About 10.sup.9 cells in a person with body weight 70 kg are enough to cause the lymphocytes to respond in the SCM test in the characteristic pattern of malignancy. In mice, when as few as 7.5.times.10.sup.5 Ehrlich ascites (tumor) cells are implanted, the pattern of the response in the SCM test is altered; response to cancer-specific antigens is induced, while the normal response to PHA is virtually eliminated. (L. Cercek and B. Cercek, "Changes in SCM-Response of Lymphocytes in Mice After Implantation with Ehrlich Ascites Cells," Europ. J. Cancer, 17, 167-171 (1981)).
By contrast, when malignancies of the breast or colon are surgically removed, the response of the surgical patients' lymphocytes to a generally cancer-associated antigen, cancer basic protein (CaBP), is lost within 24 hours after surgery. The normal response to PHA is slower to recover in these patients; that response returns within two weeks after surgery (L. Cercek and B. Cercek, "Changes in the SCM Response Ratio (RR.sub.SCM) After Surgical Removal of Malignant Tissue," Brit. J. Cancer 31, 250-251 (1975)).
For comparison of the response of lymphocytes to cancer-associated antigens and to PHA in the SCM test, an "SCM Response Ratio" can be calculated. This ratio is the ratio of the degree of fluorescence polarization obtained after stimulation with a cancer-associated antigen to the degree of fluorescence polarization obtained after stimulation with PHA. Since normal cells show a decrease in fluorescence polarization after treatment with PHA, but not with cancer-associated antigens, the response ratio for such normal cells is generally relatively high--from 1.3 to 1.6. By contrast, lymphocytes isolated from patients with malignant conditions show a much lower response ratio of 0.6 to 0.8. In some "pre-malignant" conditions such as polyposis coli and hyperkeratosis of the skin, an intermediate ratio of 0.8 to 1.0 may be seen. (Cercek, Cercek and Franklin, 1974). This suggests that the SCM test may have diagnostic value even before the cancer has reached a clinically or histologically detectable stage.
When lymphocytes from cancer patients are challenged in the SCM test with pieces of whole cancer tissue, they respond only to tissues of the same types of cancer from which the lymphocytes were derived (L. Cercek and B. Cercek, "Apparent Tumour Specificity with the SCM Test," Brit. J. Cancer 31, 252-253 (1975)). However, the purified cancer-associated antigens which are the subject of this invention do not give a response specific for a particular type of cancer when used to challenge lymphocytes in the SCM test. In other words, these purified antigens elicit a response in lymphocytes from patients with any type of cancer. As discussed previously, this property may make such antigens clinically useful for generalized screening tests. However, the response to the SCM test may fail at extremely advanced stages of malignant disease, when the cancer has already metastasized.
Among the cancer-associated antigens which are known to be effective in the SCM test are cancer basic protein (CaBP) and myelin basic protein (MBP). Cancer basic protein is a basic protein or group of similar basic proteins isolated from cancerous tumors. Myelin basic protein (MBP) is an approximately 15,000 dalton protein which is a major component of myelin. An abnormal allergic response to myelin is believed to be part of the disease process in a number of nervous system disorders, such as Guillain-Barre' syndrome and very likely multiple sclerosis. As expected, lymphocytes from multiple sclerosis respond to MBP in the SCM test; however, such lymphocytes do not respond to CaBP, and respond as do lymphocytes from normal individuals to PHA. This pattern of responses differentiates lymphocytes from patients with multiple sclerosis from lymphocytes from patients with malignant disorders.
However, neither CaBP nor MBP is ideal for use as a generalized challenging agent in the SCM test. Both must be purified from natural material, which creates the possibility of variability from batch to batch, as well as the possibility of contaminants having a profound effect on the test. CaBP, furthermore, is a relatively nonspecific antigen, and it has therefore fallen into disfavor. MBP is relatively difficult to purify, as the starting material is central nervous system material, and the protein is relatively hydrophobic and insoluble under many conditions. Furthermore, it is known that lymphocytes from patients with multiple sclerosis respond to MBP in the SCM test. Although the response of such lymphocytes can be differentiated from the response given by lymphocytes from patients with malignancies, such differentiation requires extra controls, such as stimulation of an aliquot of the lymphocytes with a mitogen, and complicates the test.
When synthetic experimental allergic encephalitogenic peptide (EAE peptide) became available, this peptide, which is a fragment comprising amino acid residues 114-112 of human MBP and having the amino acid sequence Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Arg, was tested in the SCM test and gave the expected positive response similar to MBP. This EAE was distributed commercially by Beckman Instruments.
2. Research Leading to the Invention
The research which led directly to the present invention began when the inventors, in work not published or revealed prior to this application, most unexpectedly discovered that Beckman EAE peptide further purified by anion-exchange chromatography did not produce the expected response in the SCM test when used to challenge lymphocytes from cancer patients. Thus the synthetic peptide that was expected to be useful in the SCM test proved to be a failure.
Accordingly, there is a need for a peptide useful as a substitute for cancer basic protein and myelin basic protein as a challenging material for lymphocytes from cancer cells in the SCM test. Preferably the peptide is nonspecific with respect to the type of cancer affecting the donor of the challenged lymphocytes. It is desirable that the peptide be useful for rapid screening of lymphocyte samples for cancer with the SCM test and be useful for detecting cancer in its early stages. A composition comprised of such peptides is designated an "SCM-active cancer recognition composition," or, since the recognition of cancer is the major purpose of the test and any composition must have that activity to be of value in the test, more simply as an "SCM-active composition."